Thyroid stimulating hormone (TSH) is one of family of glycoprotein hormones that consist of two non-covalently linked, dissimilar subunits, designated alpha and Beta. Detailed information is lacking concerning the cellular processes involved in the biosynthesis of the glycoprotein hormones. We propose to investigate the cellular events that take place in the biosyntheis of TSH; specificially, we wish to understand the cellular mechanisms of post-translational processing of the alpha and Beta subunits including cleavages of precursors, glycosylation, and assembly of chain. We wish to determine how these processes are regulated, how they may be interdependent, and how they may be involved in intracellular transport, packaging, and secretion of the hormone. An essential first approach to these studies will be to obtain information about the primary structure of the subunits and their precursors. To accomplish this, we propose: (a) to determine the amino acid sequence to TSH-subunits isolated from a transplantable thyrotroph tumor developed in our laboratory, and (b) to determine the primary sequences of the putative subunit precursors found by translations of thyrotroph tumor mRNA in heterologous cell-free systems. Two approaches will be used for the sequence analyses: (i) radiomicrosequencing of the protein translation products, and (ii) sequencing of cDNA made complementary to the subunit mRNAs. The combination of these two approaches will provide a more expeditious and unambiguous means of obtaining information about the primary sequences of the subunits than would one or the other approach used alone. In addition, the sequencing of the cDNA will also provide information about the structure of the non-coding regions of the mRNAs for the subunits. This information of the primary structures will be used in analyses of the processes of glycosylation and post-translation cleavages of the subunits precursors. Further, we propose to analyze by electrophoresis and by electron microscopic radioautography the migration and assembly of newly synthesized subunit chains in intact thyrotrophic cells in vitro. These initial studies will then provide the basis for subsequent studies of the cellular mechanisms involved in the regulation of subunit synthesis, assembly, and secretion mediated by thyrotrophin releasing hormone and thyroid hormones.